The hepatotoxicity of acetaminophen (A) is mediated by reactive metabolite(s). We have previously shown that this metabolite is not formed by N-hydroxylation 3,4-epoxidation, or 2,3-epoxidation. In this work we show that that there is more than one reactive metabolite, more covalent binding occurs with 14C-ring-A than with 14C-A. Acetamide was found to be a microsomal metabolite of 14C-acetyl-A. Acetamide formation plus covalent binding of 14C-acetyl-A equal 14C-ring A binding. These data indicate two reactive metabolites of A. Ascorbic acid blocked acetamide formation and covalent binding of both labels. Addition of exogeneous protein to the microsomal incubation mixture increased covalent binding and decreased acetamide formation. These data are consistent with N-acetylimidoquinone being an acetreactive metabolite of A and hydrolysis of N-acetylimidoquinone yields acetamide plus benzoquinone, which also binds to protein. One electron reduction of N-acetylimidoquinone will yield N-acetylsemiquinone which may also be a reactive metabolite. The data are consistent with the concept that A is converted to N-acetylimidoquinone, by a previously unknown mechanism for cytochrome P-450.